Biomedical subjects

R Krishna

Publications and source records attributed to R Krishna.

At least 19 recordsLinked to original sources

PubMed2006

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery.

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102-105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.

Allosteric Regulation↗

PubMed2006

Influence of isotherm inflection on the loading dependence of the diffusivities of n-hexane and n-heptane in MFI zeolite. Quasi-elastic neutron scattering experiments supplemented by molecular simulations.

Quasi-Elastic Neutron Scattering (QENS) experiments were carried out to determine (a) Fick diffusivity, D (b) self-diffusivity, Dself, and (c) 1/Gamma, the inverse of the thermodynamic correction factor, for n-hexane (nC6) and n-heptane (nC7) in MFI zeolite (all silica silicalite-1) at 300 K for a variety of loadings. These experimental results are compared with configurational-bias Monte Carlo (CBMC) and molecular dynamics (MD) simulations of, respectively, the adsorption isotherms and diffusivities. For n-hexane, the CBMC simulated isotherm shows a slight inflection at a loading=4 molecules per unit cell; this inflection manifests, also, in the loading dependence of 1/Gamma, obtained from QENS. The trend in the loading dependence of the Fick D and Dself of nC6 obtained from QENS matches the MD simulation results. For nC7 the CBMC simulated isotherm shows a strong inflection at a loading=4 molecules per unit cell. At this loading=4, 1/Gamma tends to zero and there is a very good match between QENS and molecular simulations for the loading dependence of 1/Gamma. Both MD simulations and QENS data on the Fick diffusivity shows a sharp maximum at a loading in the region of=4. For both nC6 and nC7 the simulated values of diffusivity are about an order of magnitude higher than those determined from QENS.

Journal Article↗

PubMed2005

Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2.

The structure of the double mutant K53,56M has previously been refined at 1.9 A resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 A data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states.

Animals↗

PubMed2005

Diffusion of alkane mixtures in zeolites: validating the maxwell-stefan formulation using MD simulations.

Molecular dynamics (MD) simulations have been carried out for pure components, binary, ternary, and quaternary mixtures containing methane, ethane, propane, and n-butane in FAU zeolite at 300 K for a range of molecular loadings Theta, approaching saturation limits. The n-dimensional matrix of Maxwell-Stefan (M-S) diffusivities [Delta], defined by (N) = -rho[Delta][Gamma](nabla Theta), was determined along with the self-diffusivities, D(i)(,self). Additionally, configurational-bias Monte Carlo (CBMC) simulations were carried out to obtain the pure component sorption isotherms and the saturation capacities Theta(i)(,sat). From the information on Delta(ij), D(i)(,self), and Theta(i)(,sat), the various M-S diffusivities were determined: (1) component D(i), reflecting the interactions of the species i with the zeolite, self-exchange D(ii), and (2) binary exchange D(ij). The obtained data underline the major advantage of the M-S formulation that at a given occupancy, theta = Sigma(N)(n)(i=l)Theta(j)/Theta(j)(,sat) within the zeolite, the D(i) has nearly the same value for species i whether this species is present on its own or in a mixture with other species. The same advantage holds, too, for the self-exchange D(ii); the value at a given occupancy, theta, is the same whether determined from pure component, binary, or ternary mixture data. For all binary and ternary mixtures studied, it was verified that the binary exchange coefficient D(ij) can be interpolated from the corresponding values of the self-exchange parameters D(ii) and D(jj) using a generalization of the interpolation formula developed earlier (Skoulidas et al., Langmuir, 2003, 19, 7977). We also demonstrate that if the occupancy dependence of the pure component parameters D(i) and D(ii) are modeled properly, this information is sufficient to provide very good estimates of the matrix [Delta] for mixtures with 2, 3, or 4 components over the entire range of loadings. Simulations of mixture diffusion of alkanes in MFI and LTA confirm that the above-mentioned advantages of the M-S formulation also hold for these zeolite topologies.

Journal Article↗

PubMed2004

Adsorption of fluorescein by protein crystals.

Adsorption characteristics of native and cross-linked lysozyme crystals were examined using fluorescein as model adsorbate. The adsorption isotherms exhibited Langmuir or linear behavior. The affinity constant (b1) and the adsorption capacity (Qsat) for fluorescein were found to depend on the type and concentration of co-solute present in the solution. The dynamics of adsorption isotherm transition from Langmuir to linear showed that affinity of lysozyme for solutes increases in the order 2-(cyclohexylamino)ethanesulphonic acid (CHES), 4-morpholinepropanesulphonic acid (MOPS), acetate, fluorescein. Furthermore, the crystal morphology, the degree of cross-linking of the crystals, and, in particular, solution pH were identified as factors determining fluorescein adsorption by the lysozyme crystals. These factors seem to affect crystal capacity for the solute more than affinity for the solute. Adsorption of fluorescein by cross-linked tetragonal lysozyme crystals was exponentially dependent on the lysozyme net charge calculated from the final solution pH. The 3-5-fold increase in the fluorescein adsorption as a result of cross-linking is presumably due to the increasing hydrophobicity of the lysozyme crystal.

Adsorption↗

PubMed2004

Force field parametrization through fitting on inflection points in isotherms.

We present a method to determine potential parameters in molecular simulations of confined systems through fitting on experimental isotherms with inflection points. The procedure uniquely determines the adsorbent-adsorbate interaction parameters and is very sensitive to the size parameter. The inflection points in the isotherms are often related to a subtle interplay between different adsorption sites. If a force field can predict this interplay, it also reproduces the remaining part of the isotherm correctly, i.e., the Henry coefficients and saturation loadings.

Adsorption↗

PubMed2004

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization.

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.

Biological Transport↗

PubMed2003

Crystal structures of Mycobacterium smegmatis RecA and its nucleotide complexes.

The crystal structures of Mycobacterium smegmatis RecA (RecA(Ms)) and its complexes with ADP, ATPgammaS, and dATP show that RecA(Ms) has an expanded binding site like that in Mycobacterium tuberculosis RecA, although there are small differences between the proteins in their modes of nucleotide binding. Nucleotide binding is invariably accompanied by the movement of Gln 196, which appears to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding loops. These observations provide a framework for exploring the known properties of the RecA proteins.

Adenosine Diphosphate↗

PubMed2001

In vitro protein binding studies with BMS-204352: lack of protein binding displacement interaction in human serum.

BMS-204352, a maxi-K channel opener, is currently under development for the treatment of stroke. Protein binding of BMS-204352 was determined in sera from several species, namely, rat, monkey, dog, and human. Data indicated that the compound was shown to be highly protein bound in serum from all species (ca. 99.6%). In order to test for the potential for drug-drug interactions and competitive displacement of BMS-204352 by diazepam, phenytoin, propranolol, and warfarin, in vitro experiments were performed using spiked human serum and ex vivo human plasma samples. Protein binding was determined using equilibrium dialysis for 4 h at maximal therapeutic concentrations for each drug alone or in appropriate combination in spiked serum samples. Ex vivo samples from a clinical BMS-204352 study (0, 1, and 24 h) were dialyzed separately after addition of diazepam, phenytoin, propranolol, or warfarin. Drug content in biological matrices was measured for radioactivity using liquid scintillation counting. Results indicated that (1) addition of diazepam, phenytoin, propranolol, or warfarin did not alter the free fraction of BMS-204352; (2) BMS-204352 did not displace diazepam, phenytoin, propranolol, or warfarin from their protein binding sites, and (3) comparison of ex vivo plasma samples after BMS-204352 dosing indicated no impact of BMS-204352 and/or its metabolites on the free fraction of diazepam, phenytoin, propranolol, or warfarin. In conclusion, the potential for a drug-drug interaction due to alterations in protein binding with BMS-204352 is unlikely.

Animals↗

PubMed2001

Intermediate-term outcomes of 350-mm(2) Baerveldt glaucoma implants.

OBJECTIVE: To determine the intermediate-term outcome of 350-mm(2) Baerveldt glaucoma implants. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Sixty-five patients (65 eyes). INTERVENTION: Implantation of 350-mm(2) Baerveldt glaucoma drainage device. MAIN OUTCOME MEASURES: Intraocular pressure, number of glaucoma medications, best-corrected Snellen visual acuity, length of follow-up, risk factors for failure, and complications. RESULTS: Mean intraocular pressure was reduced from a preoperative value of 32 mmHg to a 2-year postoperative value of 14 mmHg (56% reduction, P < 0.001). Success rates at 2-year follow-up were 71%, 81%, 78%, 60%, and 47% for the overall group, primary open-angle glaucoma group, neovascular group, uveitic group, and other group, respectively. After accounting for the effect of diagnosis group, significant risk factors in the overall group for failure at 2 years included younger age, high preoperative intraocular pressure, and more prior incisional surgeries. Glaucoma medication use in our overall study population was reduced from 2.5 mean preoperative medications to 0.8 postoperative medications (68%). Median change in Snellen visual acuity between preoperative and 2-year postoperative visits was not significant in the overall group. Postoperative complications included choroidal effusion in 15 patients (23%), tube obstruction by blood or vitreous in five patients (8%), phthisis in four patients (6%), aqueous misdirection in two patients (3%), strabismus in two patients (3%), tube-cornea touch in two patients (3%), endophthalmitis in two patients (3%), and retinal detachment in two patients (3%). No patients had suprachoroidal hemorrhage. CONCLUSIONS: The 350-mm(2) Baerveldt glaucoma implants are a safe and effective treatment for intermediate-term intraocular pressure control in patients with refractory glaucoma.

Adult↗

PubMed2001

Visualization of bioavailable liposomal doxorubicin using a non-perturbing confocal imaging technique.

Commonly employed tissue processing techniques can significantly alter tissue drug distribution patterns for liposomal encapsulated drugs by virtue of drug leakage via loss of membrane integrity. We report here a method that has been developed to determine the fluorescence of bioavailable doxorubicin (DOX) in tissues after administration of liposomal DOX formulations. A non-perturbing confocal fluorescence microscopy (CFM) technique with image processing analysis was used with unprocessed fresh tissues. This method takes advantage of the fact that considerable quenching occurs when DOX is within liposomes, leading to the selective visualization of the fluorescence due to DOX released from liposomes. We demonstrate that fresh tissue confocal imaging can be applied to provide detailed drug distribution information with improved accuracy and is a superior method for analyzing tissue distribution of liposome entrapped fluorescent agents.

Animals↗

PubMed2001

Molecular and pharmacological strategies to overcome multidrug resistance.

Multidrug resistance is a major obstacle to the effective treatment of cancer. Despite vast improvements in our understanding of the mechanisms of drug resistance, relatively few significant advances have been made towards effectively circumventing it in a clinical setting. The ability to modulate multidrug resistance has been complicated by the fact that many human tumors simultaneously exhibit multiple resistance mechanisms. In order to effectively overcome multidrug resistance it will be necessary to design new strategies that combine multiple modulating agents and approaches. This review provides an overview of the major causes of multidrug resistance and summarizes many of the current approaches being taken to overcome it. We also describe how liposomal drug delivery systems can be utilized to aid in achieving these goals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Modulation of P-glycoprotein (PGP) mediated multidrug resistance (MDR) using chemosensitizers: recent advances in the design of selective MDR modulators.

Over the past two decades, a number of chemical entities have been investigated in the continuing quest to reverse P-glycoprotein (PGP) mediated multidrug resistance (MDR) in cancer. The complexity of interactions between these agents and the proteins responsible for MDR in conjunction with the challenges associated with developing SAR/QSAR relationships for MDR modulators has hampered our ability to develop agents that modulate MDR with enhanced specificity of target, increased efficacy, and minimized toxicity when coadministered with anticancer drugs. With an increased understanding of the molecular interaction, target-mediated SAR and combinatorial chemistry approaches, newer more selective inhibitors have been recently reported. These agents have shown remarkable promise in preclinical trials; although their ultimate clinical therapeutic utility remains to be established. The emphasis of this review is placed on the current understanding of modulator-drug transport protein interactions and to review the advances in the structure-based design, synthetic efforts and the cellular pharmacology of MDR modulating activity of a number of known PGP inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Liposomal and nonliposomal drug pharmacokinetics after administration of liposome-encapsulated vincristine and their contribution to drug tissue distribution properties.

We have determined the pharmacokinetics of liposomal vincristine, in a Lewis lung carcinoma solid tumor model in mice, with the aim of differentiating the contribution of liposomal and nonliposomal (released from liposomes) drug pools to the overall pharmacokinetic profile. Two types of liposomal formulations were used: one composed of 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol (Chol) (55/45; mol/mol) and the other composed of sphingomyelin/cholesterol (SM/Chol; 55/45; mol/mol). Vincristine elimination from the circulation after injection of conventional, aqueous formulated vincristine (C-VINC) was characterized by a short half-life (1.36 h), low plasma area under the plasma concentration-time curve (AUC) (0.59 microg x h/ml), and large volume of distribution (145 ml). Total drug elimination from the circulation after liposomal vincristine injection using SM/Chol liposomes was characterized by a prolonged half-life (6.6 h), increased plasma AUC (213 microg x h/ml) and small volume of distribution (2.0 ml). Our results indicate that > or =98% of the total vincristine measured in the plasma of mice administered with liposomal vincristine was encapsulated within the liposomes. The systemic exposure to free drug after administration of liposomal formulations was significantly lower than that observed after the injection of C-VINC. Plasma concentrations of free drug remained between 0.025 and 0.05 microg/ml over 4 h of postinjection for both liposomal formulations. In contrast, concentrations between 0.1 and 0.35 microg/ml were observed following C-VINC administration. Free plasma drug concentrations did not correlate with vincristine tissue distribution properties following administration of liposomal vincristine formulations. Rather, accumulation of vincristine in tissues appeared to be influenced primarily by the drug retention properties of the liposome. While the reduced systemic exposure to free vincristine correlates with reduced toxicity, additional information (such as liposome drug release properties) may be necessary to correlate pharmacokinetic behavior with antitumor activity.

Animals↗

PubMed2000

Increased intracellular drug accumulation and complete chemosensitization achieved in multidrug-resistant solid tumors by co-administering valspodar (PSC 833) with sterically stabilized liposomal doxorubicin.

We have previously demonstrated that liposome encapsulation of doxorubicin (DOX) can alleviate adverse interactions with non-encapsulated DOX and the cyclosporine multidrug-resistant (MDR) modulator Valspodar. We have now investigated the behavior of different liposomal DOX formulations in MDA435LCC6/MDR-1 human breast cancer solid tumor xenograft models to identify liposome characteristics associated with enhanced therapeutic activity and the mechanism whereby increased chemosensitization is achieved. Toxicity studies incorporating conventional phosphatidylcholine (PC)/cholesterol (chol) and sterically stabilized (polyethylene glycol 2000 [PEG]-containing) formulations of DOX indicated that whereas PC/Chol DOX was approximately 3-fold more toxic in the presence of Valspodar, PEG containing distearoylglycerophosphocholine (DSPC)/Chol DOX was minimally affected. In mice bearing MDR tumors, co-administration of Valspodar and egg phosphocholine (EPC)/Chol DOX resulted in modest MDR modulation and efficacy, whereas the sterically stabilized formulation induced reductions in tumor growth equivalent to that achieved for drug-sensitive tumors treated with non-encapsulated DOX. Pharmacokinetic studies revealed a 2.5-fold increase in plasma DOX area under the curve (AUC) upon co-administration of Valspodar with EPC/Chol DOX whereas no such alterations were observed with the sterically stabilized liposomes. Compared to non-encapsulated DOX combined with Valspodar, improvements in efficacy and toxicity correlated with the extent to which liposomal DOX formulations were able to circumvent pharmacokinetic interactions. Confocal microscopy demonstrated that Valspodar increased cell-associated DOX which correlated with the level of anti-tumor efficacy.

Animals↗

PubMed2000

Baerveldt drainage implants in eyes with a preexisting scleral buckle.

OBJECTIVE: To describe the surgical insertion of a Baerveldt drainage implant and postoperative visual acuity and intraocular pressure (IOP) outcomes in patients with a preexisting scleral buckle. METHODS: Medical records of all patients with a preexisting scleral buckle who underwent insertion of a Baerveldt drainage implant at Bascom Palmer Eye Institute, Miami, Fla, from January 1, 1994, through December 31, 1998, were reviewed. Outcome measures included visual acuity and IOP at 1 year. RESULTS: At 1 year postoperatively, 14 (88%) of 16 patients had stable or improved visual acuity. Preoperatively, mean IOP was 30.9 mm Hg and the mean number of antiglaucoma medications was 3.4; at 1 year postoperatively, mean IOP was 12.0 mm Hg and the mean number of antiglaucoma medications was 0.8 (P<.001). Nine patients (56%) achieved an IOP of greater than 5 and no greater than 21 mm Hg without medication, and an additional 7 (44%) achieved this level of IOP control with medication. No patient required further surgery for uncontrolled IOP during the follow-up interval, which ranged from 19. 1 to 45.5 months. CONCLUSION: Baerveldt drainage device insertion behind or over a preexisting encircling band is often successful in managing refractory glaucoma in patients who have undergone previous scleral buckling procedures. Arch Ophthalmol. 2000;118:1509-1513

Adolescent↗

PubMed2000

Preparation of recombinant bovine, porcine, and porcine W4R/R5K leptins and comparison of their activity and immunoreactivity with ovine, chicken, and human leptins.

Recombinant ovine Ala-leptin (GenBank Accession No. U84247, of ovine leptin), previously prepared in our laboratory in prokaryotic expression plasmid pMON3401, was mutated using a mutagenesis kit to prepare plasmids encoding for bovine (GenBank Accession No. U50365) and porcine (GenBank Accession No. U59894) leptins and for porcine leptin analogue W4R/R5K. Escherichia coli cells transformed with these plasmids overexpressed large amounts of these proteins upon induction with nalidixic acid. The expressed proteins, found in inclusion bodies, were refolded and purified to homogeneity using subsequently anion- and cation-exchange chromatography. All three purified proteins showed a single band of the expected molecular mass of 16 kDa in SDS-PAGE in the presence of reducing agent and were composed of 90-100% monomers. Proper refolding was evidenced by comparing their CD spectra to those of previously prepared chicken and ovine leptins and to commercially available human leptin. The amino acid content of the purified proteins closely resembled the predicted composition. The biological activity of bovine leptin, porcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor. All three proteins, as well as ovine and chicken leptins, but not human leptin, exhibited a very high degree of cross-immunoreactivity against antiserum raised against ovine leptin in rabbits. In contrast, none or very low cross-immunoreactivity was observed against antiserum raised against ovine leptin in goats.

Amino Acid Sequence↗

PubMed2000

Utilisation of bubble resonance phenomena to improve gas-liquid contact.

In several branches of science and technology a gaseous phase is dispersed into a liquid in the form of bubbles, a gaseous component then dissolves into the liquid and subsequently undergoes chemical reaction. The overall process performance can be improved substantially when the area of gas-liquid contact is increased. By subjecting the liquid phase to low frequency vibrations, the bubbles are shown to suffer significant breakage, induced by resonance. When the vibration is properly tuned, the interfacial area is found to increase by a factor of 1.8-2.4, depending on the properties of the liquid. Resonance-induced bubble breakage phenomena have a great potential for improving the rates of chemical processes involving fast reactions, with minimal energy input.

Journal Article↗